PI (phosphatidylinositol) is a ubiquitous eukaryotic phospholipid which serves as a precursor for messenger molecules and GPI (glycosylphosphatidylinositol) anchors. PI is synthesized either de novo or by head group exchange by a PIS (PI synthase). The synthesis of GPI anchors has previously been validated both genetically and chemically as a drug target in Trypanosoma brucei, the causative parasite of African sleeping sickness. However, nothing is known about the synthesis of PI in this organism. Database mining revealed a putative TbPIS gene in the T. brucei genome and by recombinant expression and characterization it was shown to encode a catalytically active PIS, with a high specificity for myo-inositol. Immunofluorescence revealed that in T. brucei, PIS is found in both the endoplasmic reticulum and Golgi. We created a conditional double knockout of TbPIS in the bloodstream form of T. brucei, which when grown under non-permissive conditions, clearly showed that TbPIS is an essential gene. In vivo labelling of these conditional double knockout cells confirmed this result, showing a decrease in the amount of PI formed by the cells when grown under non-permissive conditions. Furthermore, quantitative and qualitative analysis by GLC-MS and ESI-MS/MS (electrospray ionization MS/MS) respectively showed a significant decrease (70%) in cellular PI, which appears to affect all major PI species equally. A consequence of this fall in PI level is a knock-on reduction in GPI biosynthesis which is essential for the parasite's survival. The results presented here show that PI synthesis is essential for bloodstream form T. brucei, and to our knowledge this is the first report of the dependence on PI synthesis of a protozoan parasite by genetic validation.